Thermo Fisher Scientific Membrane Permeability/Dead Cell Apoptosis Kit User guide

Brand: Thermo Fisher Scientific

Model: Membrane Permeability/Dead Cell Apoptosis Kit

Type: User guide

Membrane Permeability/Dead Cell Apoptosis Kit with PO‑PRO™‑1

and 7‑Aminoactinomycin D for Flow Cytometry

Catalog Number V35123

Pub.No. MAN0002438 Rev. A.0

WARNING! Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear,

clothing, and gloves. Safety Data Sheets (SDSs) are available from thermoﬁsher.com/support.

Product description

Apoptosis is a carefully regulated process of cell death that occurs as a normal part of development. Inappropriately regulated apoptosis

is implicated in disease states, such as Alzheimer’s disease and cancer. Apoptosis is distinguished from necrosis, or accidental cell death,

by characteristic morphological and biochemical changes, including compaction and fragmentation of the nuclear chromatin, shrinkage of

the cytoplasm, and loss of membrane asymmetry. Furthermore, during apoptosis the cytoplasmic membrane becomes slightly permeant.

Certain dyes, such as the violet ﬂuorescent PO‑PRO™-1 dye can enter apoptotic cells, whereas other dyes, such as the red ﬂuorescent

dye, 7-aminoactinomycin D (7-AAD), cannot. The use of YO‑PRO™-1 dye (closely related to PO‑PRO™-1 dye) with propidium iodide

provides a sensitive indicator for apoptosis. PO‑PRO™-1 dye and 7-AAD provide analogous performance. In addition, annexin V gives

poor results with trypsinized cells while PO‑PRO™-1 dye provides the same eciency with trypsinized cells as it does with suspension

cells.

The Membrane Permeability/Dead Cell Apoptosis Kit with PO‑PRO™‑1 and 7‑Aminoactinomycin D for Flow Cytometry provides a rapid

and convenient assay for apoptosis. The kit contains ready-to-use solutions of both PO‑PRO™-1 and 7-AAD dyes. After staining a cell

population with PO‑PRO™-1 dye and 7-AAD, apoptotic cells show violet ﬂuorescence, dead cells show violet and red ﬂuorescence, and

live cells show little or no ﬂuorescence (Figure1). These populations can easily be distinguished by a ﬂow cytometer that uses both a

violet laser and the 488 nm line of an argon-ion laser for excitation.

USER GUIDE

For Research Use Only. Not for use in diagnostic procedures.

Figure1Flow cytometry analysis with the Membrane Permeability/Dead Cell Apoptosis Kit with PO‑PRO™‑1 and

7‑Aminoactinomycin D for Flow Cytometry.

Jurkat cells (human T-cell leukemia) were treated with 10 µM camptothecin for 4 hours (bottom panel) or untreated (as control, top panel).

Cells were then treated with the reagents in the kit and analyzed by ﬂow cytometry using 405 nm and 488 nm excitation. Note that the

camptothecin-treated cells have a higher percentage of apoptotic cells (indicated by an “A”) than the basal level of apoptosis seen in the control

cells. L = live cells, D = dead cells.

Because no single parameter deﬁnes apoptosis in all systems, we strongly suggest using a combination of dierent measurements for

reliable detection of apoptosis. Refer to our website at http://thermoﬁsher.com/apoptosis for a wide selection of products for apoptosis

research.

Contents and storage

Component Amount Concentration Storage[1]

PO‑PRO™-1 dye

(Component A) 500µL 1mM solution in DMSO 2°C to 6°C[2]

Dessicate

Protect from light[3]

7-aminoactinomycin D (7-AAD)

(Component B) 200µL 1mg/mL solution in DMSO

Number of assays: 200 ﬂow cytometry assays containing 2 × 105 to 1 × 106 cells in a 1mL volume.

Approximate ﬂuorescence excitation/emission maxima: PO‑PRO™-1: 434/456nm; 7-AAD: 546/647nm.

[1] Kit is stable for 6months when stored as directed.

[2] For long-term storage, store the vial of PO‑PRO™-1 dye at ≤–20°C.

[3] The PO‑PRO™-1 dye and 7-AAD are light sensitive and may be handled in normal room light, but avoid prolonged exposure to light.

Before you begin

Required materials not supplied

• Samples (appropriate sample concentrations range from 2 × 105 to 1 × 106 cells/mL)

• Inducing agent

•Phosphate-buered saline (PBS)

• Deionized water

2 Membrane Permeability/Dead Cell Apoptosis Kit with PO‑PRO™‑1 and 7‑Aminoactinomycin D for Flow Cytometry User Guide

Label cells for ﬂow cytometry

The assay is optimized using Jurkat cells (human T-cell leukemia line) treated with camptothecin to induce apoptosis. Some modiﬁcations

may be required for use with other cell types.

1. Induce apoptosis in cells using the desired method. Prepare a negative control by incubating cells in the absence of inducing agent.

2. Harvest the cells after incubation, wash in cold phosphate-buered saline (PBS), and adjust the cell density to approximately

1×106 cells/mL in PBS. For each assay, use a 1mL volume.

3. Add 2.5µL PO‑PRO™-1 stock solution (Component A) and 1 µL 7-AAD stock solution (Component B) to each 1mL of cell

suspension.

4. Incubate the cells on ice for 30 minutes.

5. Immediately after incubation, analyze the stained cells by ﬂow cytometry using violet and 488 nm excitation and measuring the

ﬂuorescence emission using 440nm and 670nm bandpass ﬁlters (or their near equivalents).

The population should separate into 3 groups: Live cells will show only a low level of ﬂuorescence; apoptotic cells will show violet

ﬂuorescence; and necrotic cells will show both red and violet ﬂuorescence (see Figure1). Conﬁrm the ﬂow cytometry results by

viewing the cells under a ﬂuorescence microscope using the appropriate ﬁlters.

Limited product warranty

Life Technologies Corporation and/or its aliate(s) warrant their products as set forth in the Life Technologies' General Terms and

Conditions of Sale at www.thermoﬁsher.com/us/en/home/global/terms-and-conditions.html. If you have any questions, please

contact Life Technologies at www.thermoﬁsher.com/support.

Life Technologies Corporation | 29851 Willow Creek Road | Eugene, Oregon 97402 USA

For descriptions of symbols on product labels or product documents, go to thermoﬁsher.com/symbols-deﬁnition.

Revision history:Pub.No. MAN0002438

Revision Date Description

A.0 19 July 2022 The format and content were updated. The version numbering was reset to A.0 in conformance with internal document control.

2.00 12 March 2010 Names were changed.

1.00 17 June 2005 New document for the Membrane Permeability/Dead Cell Apoptosis Kit with PO‑PRO™‑1 and 7‑Aminoactinomycin D for Flow

Cytometry.

The information in this guide is subject to change without notice.

DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT,

PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.

Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all

applicable Limited Use Label Licenses.

thermoﬁsher.com/support|thermoﬁsher.com/askaquestion

thermoﬁsher.com

19 July 2022

Thermo Fisher Scientific Membrane Permeability/Dead Cell Apoptosis Kit User guide

Thermo Fisher Scientific Membrane Permeability/Dead Cell Apoptosis Kit User guide

Thermo Fisher Scientific Membrane Permeability/Dead Cell Apoptosis Kit User guide

Related papers

Other documents